The purpose of this project is to study the breakdown of blood glycoproteins in hepatic lysosomes. Galactose-terminating serum asialoglycoproteins are first bound via their carbohydrate chains to a galactose-specific binding protein present on the sinusoidal surface of hepatocytes. The bound asialoglycoprotein is rapidly transported in pinocytic vesicles to the lysosomes, and ultimately digested to the level of amino acids and sugars in that cellular compartment. For this current study, the degradation of (3H) glucosamine-labeled asialo-orosomucoid will be examined in a perfused rat liver in the presence of a specific beta-D-galactosidase inhibitor, beta-d-galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT). We wish to show that beta-GalMNT can prevent lysosomal digestion of the sugar moiety of orosomucoid in the perfused liver by its inactivation of lysosomal beta-D-galactosidase. The inhibitor (0.1 mM) will be added to the perfusate 1 hour before (3H) glucosamine-labeled asialo-orosomucoid. At time intervals over a 2-hour period treated livers will be removed from perfusion, homogenized and subjected to subcellular fractionation. For each liver the amount of its beta-D-galactosidase will be assayed and the subcellular distribution and molecular weight nature of its radioactivity will be measured. The molecular size of the lysosomal radioactive material will be analyzed by column chromatography on Biogel P10, and determination of the terminal sugars on the large glycoprotein fragments will be done by reacting them with either jack bean beta-D-hexosaminidase alone or this glycosidase in combination with beta-D-galactosidase. If both enzymes must be used to cause release of N-acetyl-D-(3H)glucosamine, the terminal galactosyl residues must not have been hydrolyzed in situ. Such a result will verify that beta-D-galactosidase was inactivated. These studies with beta-GalMNT may allow us to develop an experimental tissue model for a lysosomal storage disease in which beta-galactosidase is deficient.